The Kinetics of the Enzymatic Hydrolysis of Hyaluronic Acid

The wide-spread natural occurrence of the enzyme hyaluronidase has been recognized for a number of years.The recent demonstration of the possible rale of testicular hyaluronidase in fertilization (l-3), the inhibition of hyaluronidase by salicylates (4, 5), and the presence of a hyaluronidase inhibitor in human blood have focused attention on this enzyme (6, 7).Relatively little attention has been paid to the kinetics of the reaction catalyzed by hyaluronidase.It is the purpose of this communication to present evidence that, within the usual assay range, this reaction is first order with respect to hyaluronic acid, with changing rate due to changing substrate concentration.A variety of different methods have been devised for the assay of hyaluronidase.These have been discussed in the accompanying paper (8).The viscosity method was first studied in detail by Madinaveitia and Quibbel (9).These workers found that the time required for half reduction in viscosity (half life time) is independent of substrate concentration and inversely proportional to enzyme concentration.This has since been confirmed by McClean (10) and Haas (6).The viscosity reduction method depends on the rate of decrease in the viscosity of a solution of hyaluronic acid.It has been cu&omary to determine the hyaluronidase activity in terms of the reciprocal (or some function thereof) of the half life time.Recently Swyer and Emmens (11) have reinvestigated the relationship of activity and substrate concentration and have found that at lower concentrations of substrate the activity varies with substrat,e concentration.These workers have also raised objections to the expression of activity as a reciprocal of the half life t,ime and suggested an alternate procedure which depends on the relationship of the log of concentration of enzyme and the viscosity at a specific time.When the fall in viscosity is plotted against time, a characteristic curve is obtained.It has been assumed by some that this represents a. first order react,ion.Thus JVcrthessen et crl.( 12) have used this assumption in determining the hyaluronidase content of human semen.' one plots log C a,gainst t, a st,ra.ightline is not obt.aincd;thus doubt is cast on the validity of the application of the equation for a first order reaction.